Unlike most solid cancers, Testicular Germ Cell Tumors (TGCTs) typically are highly responsive to conventional chemotherapeutics. The long-term objectives of the proposed studies are to elucidate the molecular basis for TGCT chemosensitivity and to determine whether knowledge of the underlying mechanisms can be used to sensitize other cancers that are resistant to the same therapies. We hypothesize that the sensitivity of TGCTs to conventional chemotherapy derives from unique features of the cellular DNA damage response (DDR) in germ cells, the cells from which TGCTs arise. The DDR protects cells against genome damage and in many cell types becomes activated in response to oncogenic events. This provides a barrier to tumor progression, but also creates selective pressure for DDR gene mutations, resulting in advanced cancers that harbor a dysfunctional DDR and therefore are unable to respond to genotoxic chemotherapeutics. By contrast, DDR activation is not observed in early-stage TGCTs, and testicular cancers rarely contain DDR gene mutations. We will test the hypothesis that these unique DDR features underlie the chemosensitivity of TGCTs using a novel mouse TGCT model in which the animals develop metastatic teratocarcinoma, a mixed germ cell tumor composed of teratoma and embryonal carcinoma, the latter consisting of malignant pluripotent stem cells that are similar to embryonic stem cells. While current dogma suggests that many cancers contain a stem cell population that can survive chemotherapy, resulting in relapse, we propose that TGCTs can be completely cured by chemotherapy due to the DDR-mediated hypersensitivity of the pluripotent germ cells within these cancers. In Aim 1, we will use our novel mouse TGCT model to elucidate the cellular and molecular effects of conventional chemotherapeutics on embryonal carcinoma cells in vivo and will identify genetic determinants of chemosensitivity in these cancers. In Aim 2, we will directly test whether the pluripotent stem cells in TGCTs are unique in terms of the occurrence of DNA damage following oncogenic events or in the mechanisms of DDR activation in response to oncogene-induced DNA lesions. Together, these experiments will reveal how the DDR properties of TGCTs influence oncogenic transformation as well as the degree of treatment sensitivity in advanced cancers. Understanding the molecular basis for TGCT chemosensitivity will likely provide new biomarkers for predicting therapeutic responses in a variety of cancers and yield new molecular targets for enhancing the efficacy of conventional chemotherapeutics.